U.S. patent number 6,087,117 [Application Number 08/475,684] was granted by the patent office on 2000-07-11 for production and use of human nm23 protein and antibodies therefor.
This patent grant is currently assigned to The United States of America as represented by the Department of Health. Invention is credited to Charles Richter King, Lance A. Liotta, Patricia Schriver Steeg.
United States Patent |
6,087,117 |
King , et al. |
July 11, 2000 |
Production and use of human nm23 protein and antibodies
therefor
Abstract
Human nm23 DNA and protein is disclosed as well as antibodies
which recognize human nm23 protein. The DNA and antibodies may be
used to detect nm23 in human tumors to predict the malignancy
potential of such tumors.
Inventors: |
King; Charles Richter
(Washington, DC), Steeg; Patricia Schriver (Ellicott City,
MD), Liotta; Lance A. (Potomac, MD) |
Assignee: |
The United States of America as
represented by the Department of Health (Washington,
DC)
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Family
ID: |
23676440 |
Appl.
No.: |
08/475,684 |
Filed: |
June 7, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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806932 |
Dec 11, 1991 |
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422801 |
Oct 18, 1989 |
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Current U.S.
Class: |
435/7.23;
435/7.1; 436/64; 530/387.7; 530/388.8 |
Current CPC
Class: |
C12N
9/12 (20130101); C07K 16/30 (20130101) |
Current International
Class: |
C07K
16/30 (20060101); C07K 16/18 (20060101); G01N
033/574 (); G01N 033/53 (); G01N 033/48 (); C07K
016/00 () |
Field of
Search: |
;530/387.4,350,387.7,388.8 ;435/7.1,7.23 ;436/64 |
References Cited
[Referenced By]
U.S. Patent Documents
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Re35097 |
November 1995 |
Steeg et al. |
4677058 |
June 1987 |
Tryggvason et al. |
4683195 |
July 1987 |
Mullis et al. |
4808528 |
February 1989 |
Tryggvason et al. |
4816400 |
March 1989 |
Tryggvason et al. |
5049662 |
September 1991 |
Steeg et al. |
5288852 |
February 1994 |
Yamada et al. |
|
Foreign Patent Documents
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0 303 233 |
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Aug 1987 |
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EP |
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WO 86/03226 |
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Jun 1986 |
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WO |
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WO 91/06664 |
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May 1991 |
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WO |
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WO 91/06671 |
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May 1991 |
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WO |
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Other References
Paul, W.E. "Fundamental Immunology" Second Ed. Raven Press, NY, p.
176, 1989. .
Barnes, et al. "Rapid Communication: Low nm23 Protein Expression in
Infiltrating Ductal Breast Carcinomas Correlates with Reduced
Patient Survival," Am. J. Pathol. 139(2): 245-50 (1991). .
Bevilacqua, et al. "Association of Low nm23 RNA Levels in Human
Primary Infiltrating Ductal Breast Carcinomas with Lymph Node
Involvement and Other Histopathological Indicators of High
Metastatic Potential," J. Cancer Research 49: 5185-90 (Sep. 1989).
.
Biggs, et al. "A Drosophila Gene That Is Homologous to a Mammalian
Gene Associated with Tumor Metastasis Codes for a Nucleoside
Diphosphate Kinase," Cell 933-940 (1990). .
Gilles, et al. "Nucleoside Diphosphate Kinase from Human
Erythrocytes: Structural Characterization of the Two Polypeptide
Chains Responsible For Heterogeneity of the Hexameric Enzyme," J.
Biol. Chem. 266(14): 8784-89 (1991). .
Rosengard, et al. "Reduced Nm23/Awd protein in tumor metastasis and
aberrant Drosophila Development," Nature 342: 177-80 (Nov. 1989).
.
Stahl, et al. "Identification of a Second Human nm 23 Gene.
nm23-H2," Can. Res. 51: 445 (1991). .
Steeg, et al. "Evidence for a Novel Gene Associated with Low Tumor
Metastatic Potential," J. NCI 80(3):200-4 (Apr. 1988). .
Presecan, et al. "Nucleoside diphosphate kinase from human
erythrocytes: purification, molecular mass and subunit structure,"
FEBS Letters 250:(2): 629-31 (Jul. 1989). .
Steeg, et al. Proceedings of the AACR, Symposium 11 31:504-05 (Mar.
1990) Abstract. .
Koyama et al J Biochem 95:925-935, 1984. .
Lam et al Biochem Pharmacology 35(24):4449-55, 1986. .
FEBS Lett. Yokoyama et al 206(2):287-91, 1986. .
Dulido-Cejudo et al FASEB J 3(3): A331, 1989. .
Hailat et al J. Clin. Invest 88;341-345, 1991. .
Schubart J. Biol Chem 263(24):12156-12160, 1988. .
Steeg, P.S. et al. Journal of the National Cancer Institute.
80:200-204, Apr. 1988. .
Kipps, T.J. et al. in "Handbook of Experimental Immunology" D.M.
Weir, Ed. Blackwell Scientific Publications, Oxford, England. vol.
4 Chapter 108. pp. 108.1-108.9, 1986. .
Rouse, R.V. et al. in "Handbood of Experimental Immunology" D.M.
Weir, Ed. Blackwell Scientific Publications. Oxford, England. vol.
4 Chapter 116. pp. 116.1-116.10, 1986..
|
Primary Examiner: Huff; Sheela
Assistant Examiner: Eyler; Yvonne
Attorney, Agent or Firm: Needle & Rosenberg, P.C.
Parent Case Text
The present invention is a division of application Ser. No.
07/806,932, filed Dec. 11, 1991, which is a continuation-in-part of
application Ser. No. 07/422,801 filed Oct. 18, 1989 and now
abandoned.
Claims
What is claimed is:
1. An isolated nm23 antibody which recognizes human nm23 protein
having an amino acid sequence selected from the group consisting of
the amino acid sequence designated as nm23-H1 in FIG. 6 and the
amino acid sequence designated as nm23-H2S in FIG. 6.
2. The antibody of claim 1, wherein the antibody is a monoclonal
antibody.
3. The antibody of claim 1, wherein the antibody recognizes human
nm23 protein having the amino acid sequence designated as nm23-H1
in FIG. 6.
4. The antibody of claim 3, wherein the antibody is a monoclonal
antibody.
5. The antibody of claim 1, wherein the antibody recognizes human
nm23 protein having the amino acid sequence designated as nm23-H2S
in FIG. 6.
6. The antibody of claim 5, wherein the antibody is a monoclonal
antibody.
7. The antibody of claim 3, wherein the antibody is elicited in
response to a peptide consisting of amino acids 86-102 of the amino
acid sequence designated as nm23-H1 in FIG. 6.
8. The antibody of claim 7, wherein the antibody is a monoclonal
antibody.
9. The antibody of claim 3, wherein the antibody is elicited in
response to a peptide consisting of amino acids 45-61 of the amino
acid sequence designated as nm23-H1 in FIG. 6.
10. The antibody of claim 9, wherein the antibody is a monoclonal
antibody.
11. A method of detecting human nm23 protein in a sample,
comprising:
a) contacting the sample with the antibody of claim 1 under
conditions whereby a protein/antibody immunocomplex can form;
and
b) detecting the formation of an immunocomplex, whereby the
detection of an immunocomplex indicates the detection of human nm23
protein in the sample.
12. The method of claim 11, wherein the antibody is a monoclonal
antibody.
13. The method of claim 11, wherein the antibody is elicited in
response to a peptide consisting of amino acids 86-102 of the amino
acid sequence designated as nm23-H1 in FIG. 6.
14. The method of claim 13, wherein the antibody is a monoclonal
antibody.
15. The method of claim 11, wherein the antibody is elicited in
response to a peptide consisting of amino acids 45-61 of the amino
acid sequence designated as nm23-H1 in FIG. 6.
16. The method of claim 15, wherein the antibody is a monoclonal
antibody.
17. A method of quantitating the amount of human nm23 protein in a
sample, comprising:
a) contacting the sample with the antibody of claim 1 under
conditions whereby a protein/antibody immunocomplex can form;
and
b) quantitating the amount of immunocomplex formation, whereby the
amount of immunocomplex formation indicates the amount of human
nm23 protein in the sample.
18. The method of claim 17, wherein the sample is a tumor.
19. The method of claim 17, wherein the antibody is a monoclonal
antibody.
20. The method of claim 17, wherein the antibody is elicited in
response to a peptide consisting of amino acids 86-102 of the amino
acid sequence designated as nm23-H1 in FIG. 6.
21. The method of claim 20, wherein the antibody is a monoclonal
antibody.
22. The method of claim 17, wherein the antibody is elicited in
response to a peptide consisting of amino acids 45-61 of the amino
acid sequence designated as nm23-H1 in FIG. 6.
23. The method of claim 22, wherein the antibody is a monoclonal
antibody.
24. A method of identifying a tumor with increased ability to
metastasize, comprising:
a) quantitating the amount of human nm23 protein in the tumor
according to the method of claim 17; and
b) quantitating the amount of human nm23 protein in normal cells
according to the method of claim 17, whereby an amount of human
nm23 protein in a tumor which is lower than an amount of human nm23
protein in normal cells indicates a low amount of human nm23
protein in the tumor and a low amount of human nm23 protein in the
tumor identifies a tumor with an increased ability to
metastasize.
25. The method of claim 24, wherein the human nm23 protein is
quantitated according to the method of claim 19.
26. The method of claim 24, wherein the human nm23 protein is
quantitated according to the method of claim 20.
27. The method of claim 24, wherein the human nm23 protein is
quantitated according to the method of claim 21.
28. The method of claim 24, wherein the human nm23 protein is
quantitated according to the method of claim 22.
29. The method of claim 24, wherein the human nm23 protein is
quantitated according to the method of claim 23.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to human nm23 protein, DNA encoding
human nm23 proteins, (or fragments or analogues of such DNA),
antibodies which recognize human nm23 protein and processes and
products for producing and using such materials.
2. Background Art
Steeg et al., Journal of the National Cancer Institute 80:200-204,
1988 discloses a murine, nm23 gene, and corresponding protein which
is associated with murine tumor metastatic potential.
SUMMARY OF THE INVENTION
Applicant has provided a human gene(s) encoding for a human nm23
protein(s), as well as the protein(s) and antibodies which can be
used as an aid in predicting the aggressiveness of human
tumors.
More specifically, the present invention relates to genetic testing
for cancer susceptibility, diagnosis and prognosis. The present
invention makes use of the marker of the nm23 genes, for which the
human pnm23-H1 and pnm23-H2S and murine 23 and pnm23-1 recombinant
cDNA clones have been described (3, 21-22). The genetic marker
itself can be a whole gene, a fragment thereof, a genomic or cDNA
clone, an adjacent region, or a regulatory region thereof. The
purpose of this invention is to provide novel genetic methods for
the detection of (a) susceptibility to cancer and (b) cancer
tumorgenic and metastatic potential.
These methods are based on (a) structural and sequential evaluation
of nm23 DNA and; (b) evaluation of novel nm23 expression patterns,
either at the RNA, mRNA and/or protein levels. Such information is
critical to the physician's selection of diagnostic and therapeutic
regimens for the patient, both prior to the development of cancer
and during cancer detection and treatment.
Therefore, in accordance with one aspect of the present invention,
there is provided DNA, or a fragment, analogue or derivative of
such DNA, which encodes a human nm23 protein.
In accordance with another aspect of the present invention, there
is provided a cloning or expression vehicle which includes DNA, or
a fragment, analogue or derivative of such DNA, which encodes a
human nm23 protein.
In accordance with a further aspect of the present invention, there
is provided a host; in particular cells, genetically engineered to
include DNA, or a fragment or analogue or derivative of such DNA,
which encodes a human nm23 protein or a fragment or analogue or
derivative of such DNA; i.e., such cells are modified to include
human nm23 DNA.
In accordance with yet another aspect of the present invention,
there is provided a human nm23 protein.
In accordance with yet a further aspect of the present invention,
there is provided antibodies which recognize human nm23
protein.
In accordance with still a further aspect of the present invention,
there is provided procedures for using the aforementioned DNA and
antibodies for predicting the metastasic potential of tumors.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. The bacteriophage lambda PL promoter location is indicated
by an arrow and "PL." The lambda bacteriophage lambda N gene is
indicated by a box and "N gene." The position of a ribosome binding
sequence is indicated by S/D. The position of the protein initiator
atg is indicated by "atg." Where two arrows converge, a ligation
reaction is performed. A=Ava I; R=Eco RI; B=Bam HI; Bx=Bst XI;
H=Hpa I; N=Nco I.
FIGS. 2A, 2B and 2C. Differential survival of patients with
different levels of nm23 expression. FIGS. 2A, 2B and 2C represent
scoring of 44 patients' nm23 levels by immunochemistry as
described. FIGS. 2A, 2B and 2C show results obtained by three
independent pathologists. The dotted lines are high nm23-expressing
patients and solid lines are low nm23-expressing patients. The
plotting method is of Kaplan and Meyer (8).
FIGS. 3A, 3B and 3C. In situ hybridization of pnm23-H1 to metaphase
chromosomes. FIG. 3A: Metaphase spread containing a grain on
chromosome 17 (broad arrow). Two small arrows identify nonspecific
hybridization. FIG. 3B: The same metaphase spread as shown in FIG.
3A after chromosome banding. FIG. 3C: Distribution of grains on
chromosome 17 in 61 metaphase spreads. Twenty-six (17.8%) of 146
total grains were present on chromosome 17 and twelve (46%) of
these 26 grains were localized to the 17p11-q11 region.
FIGS. 4A, 4B and 4C. Allelic deletion of nm23-H1 in tumors.
Southern hybridization to Bg1 II-digested human chromosomal DNA
samples. FIG. 4A: normal lymphocyte and breast carcinoma. FIG. 4B:
normal lung and non-small cell lung carcinoma. FIG. 4C: normal
kidney, kidney carcinoma and kidney carcinoma cell line. The Bg1 II
restriction digest identified two allelic bands at 7.6 and 2.3 kb.
A non allelic 21 kb constant band was also evident. N: normal; T:
tumor; C: tumor cell line. Numbers on the left in each panel
indicate the size of the allelic bands. Arrows on the right
indicate missing allele.
FIG. 5. Homozygous deletion of nm23-H1 in colon carcinoma
metastasis. Panel A: Southern hybridization of chromosomal DNA from
normal colonic mucosa (N), colon carcinoma (T) and lymph node
metastasis (M) to nm23-H1. Panel B: Control southern hybridization
of the same filter to human Ha-ras. The probe to Ha-ras, which maps
to chromosome 11p15.5, was a 6.6 kb insert spanning the entire
genomic sequence.
FIGS. 6A and 6B. The nucleotide and predicted amino acid sequences
of nm23-H1 and nm23-H2S cDNA clones. The asterisk marks the extent
of initial incomplete nm-23H2 cDNA clone used to isolate the
complete nm23-H2S cDNA clone.
FIG. 7. Southern blots of human rodent somatic cell hybrid DNA
samples were hybridized to a 756 bp fragment of the pnm23-H1 clone.
A 4.6 kb nm23-H1 band was allelic with 2.2 and 2.4 kb bands. A 21
kb non allelic nm23-H1 band was also detected. These bands were
readily resolved from 3.2 kb, 5.1 kb, 6.1 kb, 6.9 kb, and 7.9 kb
bands or 1.3 kb, 3.5 kb, 4.3 kb, 4.9 kb, and 5.8 kb
cross-hybridizing bands in Chinese hamster and mouse digests,
respectively. Detection of the human bands was correlated with the
presence or absence of each human chromosome in the group of
somatic cell hybrids. Discordancy represents presence of the gene
in the absence of the chromosome (.+-.) or absence of the gene
despite the presence of the chromosome (.+-.), and the sum of these
numbers divided by total hybrids examined (.times.100) represents
percent discordancy. The human-hamster hybrids contained 27 primary
clones and 13 subclones (16 positive of 40 total) and the
human-mouse hybrids represented 16 primary clones and 36 subclones
(38 positive of 52 total).
DETAILED DESCRIPTION OF THE INVENTION
The term "human nm23 gene or DNA" as used herein means a gene or
DNA which encodes a human nm23 protein, or an analogue or
derivative or fragment of such DNA or gene which encompasses or
includes a DNA sequence unique to DNA which encodes a human nm23
protein. Thus, the term human nm23 gene encompasses the genes or
DNAs of Table 1 or fragments or derivatives or analogues of such
genes.
"Human nm23 protein" means nm23 protein found in humans, or a
fragment, analogue or derivative thereof which encompasses or
includes an amino acid sequence which is unique to human nm23
protein and which preferably elicits an antibody which is
recognized by human nm23 protein. The term human nm23 protein
encompasses the proteins encoded by the genes of FIG. 6.
The term "antibody" as used herein encompasses polyclonal and
monoclonal antibodies.
The term "nm23 antibody" means an antibody which is elicited in
response to human nm23 protein or which recognizes human nm23
protein. An antibody which recognizes human nm23 protein may or may
not be elicited in response to human nm23 protein.
Applicant has presently found two different and distinct human
genes (DNA) which encode for two different and distinct nm23
proteins. The first gene is referred to herein as nm23-H1. The
second gene is referred to herein as nm23-H2S. The gene sequences
for both are shown in FIG. 6.
Although Applicant has presently identified two distinct human
genes encoding for two different nm23 proteins, the scope of the
present invention is not limited to such specific genes.
Human nm23 DNA (RNA) can be used as a diagnostic tool for detecting
and/or determining RNA or DNA. For example, such DNA or RNA may be
employed to detect mRNA expression in cancer cells to thereby aid
in predicting the malignant potential of a human tumor. The methods
which may be used include:
(1) RNA ("Northern") blotting. RNA can be isolated from tumor
samples by any of a number of standard procedures. For example,
refinements of the method of Lehrach (1) can be used. RNA is
subjected to denaturing gel electrophoresis and transferred to
nitrocellulose or other support matrix. The nm23 mRNA can be
detected by hybridization of radioactively or non-radioactively
labelled nm23-H1 or nm23-H2S. mRNA in the tumor will be reflected
by the intensity of hybridization. For comparison, hybridization
with control probes for mRNA whose level is constant (e.g. B-actin)
allows normalization of results. Detection of low levels of nm23-H1
or nm23-H2S indicates a tumor of high malignant potential.
(2) Nuclease protection assays. RNA isolated from tumor samples can
be analyzed for the content of nm23-H1 or nm23-H2S by its ability
for duplexes with a labelled complementary DNA or RNA. Using the
whole or part of the nm23-H1 or nm23-H2S nucleotide sequence,
plasmids can be generated for the production of nucleic acid probes
complementary to the corresponding mRNA. Examples of such vectors
are those based on the T7 or SP6 promoter for RNA probes or m13
phage for preparation of DNA probes using oligonucleotide priming.
Probes prepared from such vectors will be allowed to hybridize to
completion to RNA from tumor samples under conditions of excess of
probe. Either RNase can be used to remove molar unhybridized RNA
probes or S1 nuclease, or other single-stranded specific DNase, can
be used to remove unhybridized DNA probe. These are then subject to
denaturing gel electrophoresis and autoradiography. The intensity
of bands corresponding to protected probe is a measure of the
amount of either nm23-H1 or nm23-H2S from the sample. Inclusion of
nuclease protection experiments for mRNAs whose levels do not
change will allow normalization of results. Detection of tumors
with relatively low levels of nm23-H1 or nm23-H2S indicates tumors
of high malignant potential.
(2) In situ hybridization of nm23-H1 or nm23-H2S in tumor sections
allow analysis of the quantity of nm23-H1 or H2S mRNA in individual
cells of a tumor. Probe complementary to the nm23-H1 or H2S
sequence can be prepared as described above and allowed to
hybridize to mRNA within thin section of tumor sample (either
embedded by standard techniques such as by the use of paraffin, or
otherwise preserved). Unhybridized probe can be removed by
nuclease. Hybridization can be detected by autoradiography or other
methods. The intensity of hybridization reflects the amount of
nm23-H1 or H2S mRNA within the cells of the tumor. When tumor cells
contain low levels of nm23 they are likely to be highly
malignant.
Susceptibility to early onset, familial breast cancer or other
cancers could be detected by several methods, including analysis of
the inheritance pattern of allelic fragments of a nm23 gene.
Inheritance of an allele associated with the development of breast
cancer in a patient's family would signify high risk for eventual
cancer development. A second method to determine cancer
susceptibility is to determine the DNA sequence of an nm23 gene, or
its regulatory sequences, in DNA extracted from the patient's
normal tissue. The presence of a mutation in the nm23 gene which
would alter its amino acid sequence from the normal sequence would
signify high risk of cancer development. Other mutations occuring
in the regulatory DNA regions for nm23, or in intron regions
responsible for normal processing and expression of these genes
would also indicate high risk of cancer development.
Therefore, human nm23 DNA (RNA) may also be used to detect
abnormalities of such DNA in normal or cancer cells to thereby aid
in predicting the genetic predisposition for developing cancer or
the aggressiveness of the cancer (abnormalities are found in more
aggressive cells). Such methods include:
(1) DNA isolated from cells can be examined for abnormalities of
the nm23-H1 or H2S gene by blot hybridization. DNA isolated from
normal tissue and tumor tissue can be fragmented by restriction
enzymes, subjected to gel electrophoresis transferred to
nitrocellulose or other support matrix and the nm23-H1 and H2S
genes' fragments detected by hybridization using probes containing
all or part of the cDNAs described above or other regions of the
nm23-H1 or H2S gene (Southern blot procedure). Differences in
hybridization pattern between DNA from normal or tumor cells
indicate abnormalities in the nm23-H1 or H2S gene.
(2) Identification of allele loss for the nm23-H1 or H2S genes.
Restriction length polymorphisms (RFLP) for each nm23 gene can be
identified by Southern blot procedure. An RFLP may be used to
identify individual alleles for a gene in patients who are
heterozygous for an RFLP. If DNA from normal and tumor cells from a
single patient indicates that there is a an allelic loss in the
tumor DNA for either nm23-H1 or H2S, such alteration indicates a
tumor of high malignant potential.
Although the scope of the present invention is not intended to be
limited to any theoretical reasoning, there are several theories
which may explain the somatic allelic deletion of a
metastasis-associated gene in primary tumors: (a) nm23-H1 may
contribute to some aspects of the tumorigenesis process as well as
metastasis. These theories are consistent with the experimental
observations (19) that stable murine nm23-H1 transfected murine
melanoma cells exhibited a reduced incidence of primary tumor
formation; and (b) altered regulation of nm23-H1 may be an early
event in the metastatic cascade, observable in primary tumor
cells.
Additionally, Kerbel, et al. (20) have reported that occasional
metastatic cells in a primary tumor have a selective growth
advantage and at the later stages of primary tumor growth dominate
the primary tumor. This "clonal dominance" of metastatic cells may
contribute to the ability to detect nm23-H1 allelic deletions in
certain primary tumor cells. Taken together the data identify
nm23-H1 as a novel locus for allelic deletion in a variety of human
carcinomas. The allelic deletion and homozygous deletion of nm23-H1
demonstrate that this gene shares a mechanism of altered regulation
in cancer with known suppressor genes.
(3) Identification of genetic abnormalities within the gene
sequence for the nm23-H1 or H2S. Nucleotide sequence analysis can
be used to determine the gene structure of nm23-H1 or H2S in a
tumor sample. The nucleotide sequence of nm23-H1 and H2S defines a
normal sequence. Changes from these sequences in the DNA of
patients indicate tumors of high metastatic potential or the
predisposition to develop cancer.
The existence of point mutations can also be of prognostic utility
in the determination of metastatic potential. The DNA sequence of a
NM23 gene can be determined by standard methods such as dideoxy or
Maxam and Gilbert sequencing and compared to the normal NM23
sequence. Alterations which would result in a change in amino acid
sequence would be indicative of increased metastatic potential.
Alterations in the sequence of chromosomal regulatory regions for
the processing and expression of NM23 gene would also signify high
metastatic potential.
Human nm23 DNA may be incorporated into a suitable expression
vehicle to produce human nm23 protein.
The appropriate DNA sequence may be included in any of a wide
variety of vectors or plasmids. Such vectors include chromosomal,
nonchromosonal and synthetic DNA sequences; e.g., derivatives of
SV40; bacterial plasmids; phage DNA's; yeast plasmids; vectors
derived from combinations of plasmids and phage DNAs, viral DNA
such as vaccinia, adenovirus, fowl pox, virus, pseudorabies,
etc.
The appropriate DNA sequence may be inserted into the vector by a
variety of procedures. In general, the DNA sequence is inserted
into an appropriate restriction endonuclease site by procedures
known in the art. Such procedures and others are deemed to be
within the scope of those skilled in the art.
The DNA sequence in the vector is operatively linked to an
appropriate expression control sequence(s) (promoter) to direct
mRNA synthesis. As representative examples of such promoters, there
may be mentioned: LTR or SV40 promoter, the E.coli lac or trp, the
phage lambda PL promoter and other promoters known to control
expression of genes in prokaryotic and eukaryotic cells or their
viruses. The expression vector also contains a ribosome binding
site for translation initiation and a transcription terminator. The
vector may also include appropriate sequences for amplifying
expression.
In addition, the expression vectors preferably contain a gene to
provide a phenotypic trait for selection of transformed host cells
such as dihydrofolate reductase or neomycin resistance for
eukaryotic cell culture, or such as tetracycline or ampicillin
resistance in E.coli.
The vector containing the appropriate DNA sequence as hereinabove
described, as well as an appropriate promoter or control sequence,
may be employed to transform an appropriate host to permit the host
to express the protein. As representative examples of appropriate
hosts, there may be mentioned: bacterial cells, such as E.coli,
Salmonella typhimurium, fungal cells, such as yeast; animal cells
such as CHO or Bowes melanoma; plant cells, etc. The selection of
an appropriate host is deemed to be within
the scope of those skilled in the art from the teachings
herein.
It is also possible to produce human nm23 protein by conventional
peptide chemistry; e.g. by use of a peptide synthesizer and solid
phase techniques.
Human nm23 protein can be employed to produce nm23 antibodies.
Antibodies against human nm23 protein may be produced by procedures
generally known in the art. For example, polyclonal antibodies may
be produced by injecting the protein alone or coupled to a suitable
protein into a non-human animal. After an appropriate period, the
animal is bled, sera recovered and purified by techniques known in
the art. Monoclonal antibodies may be prepared, for example, by the
Kohler-Millstein (2) technique involving fusion of an immune
B-lymphocyte to myeloma cells. For example, antigen as described
above can be injected into mice as described above until a
polyclonal antibody response is detected in the mouse's serum. The
mouse can be boosted again, its spleen removed and fusion with
myeloma conducted according to a variety of methods. The individual
surviving hybridoma cells are tested for the secretion of anti-nm23
antibodies first by their ability to bind the immunizing antigen
and then by their ability to immunoprecipitate nm23-H1 and H2S from
cells. Thus, the antibody elicited in response to human nm23
protein may be either a polyclonal or a monoclonal antibody.
nm23 antibodies can be used to detect tumors which have low levels
of nm23 protein and thus an increased ability to metastasize or be
malignant. Such antibodies may or may not be purified. The format
for such assays includes:
(1) Immunohistochemical analysis. Sections of the tumor can be
reacted with anti-nm23-H1 or H2S antibodies and immunocomplexes
detected by standard and commercial approaches such as peroxidase
labelled second antibodies. The density of such immunostaining
allows an estimation of the amount of nm23-H1 or H2S produced in
the cell.
(2) Solid phase immunoassays. Such assays can be used to
quantitatively determine the amount of nm23-H1 and H2S in a soluble
extract of a tumor tissue. In such an assay one component either
antibody or antigen is fixed to a solid support.
Thus, in accordance with a further aspect of the present invention,
there is provided an assay for detection or determination of human
nm23 protein which employs nm23 antibody, of the type hereinabove
described, as a specific binder in the assay.
The assay technique which is employed is preferably an assay
wherein the nm23 antibody is supported on a solid support, as a
binder, to bind human nm23 protein present in a sample, with the
bound protein then being determined by use of an appropriate
tracer.
The tracer is comprised of a ligand labeled with a detectable
label. The ligand is one which is immunologically bound by the
human nm23 protein and such ligand may be labeled by techniques
known in the art.
Thus, for example, the human nm23 protein bound to the nm23
antibody on the solid support may be determined by the use of nm23
antibody which is labeled with an appropriate detectable label.
In such a sandwich assay technique, the labeled nm23 antibody may
be a monoclonal antibody or a polyclonal antibody; e.g. the
polyclonal antibody may be an antibody which is specific for human
nm23 protein which antibody may be produced by procedures known in
the art; for example innoculating an appropriate animal with human
nm23 protein.
The detectable label may be any of a wide variety of detectable
labels, including, enzymes, radioactive labels, chromogens
(including both fluorescent and/or absorbing dyes) and the like.
The selection of a detectable label is deemed to be within the
scope of those skilled in the art from teachings herein.
The solid support for the nm23 antibody may be any one of a wide
variety of solid supports and the selection of a suitable support
is deemed to be within the scope of those skilled in the art from
the teachings herein. For example, the solid support may be a
microtiter plate; a tube, a particle, etc.; however, the scope of
the invention is not limited to any representative support. The
nm23 antibody may be supported on the support by techniques known
in the art; e.g., by coating; covalent coupling, etc. The selection
of a suitable technique is deemed to be within the scope of those
skilled in the art from the teachings herein.
The sandwich assay may be accomplished by various techniques; e.g.,
"forward"; reverse"; or "simultaneous"; however, the forward
technique is preferred.
In a typical procedure, the nm23 antibody, which is supported on a
solid support is initially contacted with a sample containing or
suspected of containing human nm23 protein to bind specifically any
of such protein present in the sample to such antibody on the
support.
After washing of the solid support, the support is contacted with a
tracer which binds to human nm23 protein. If such protein is
present in the sample, the tracer becomes bound to such protein
bound to the antibody on the solid support, and the presence of
tracer on the solid support is indicative of the presence of human
nm23 protein in the sample. The presence of tracer may be
determined by determining the presence of the detectable label by
procedures known in the art.
Although the preferred procedure is a sandwich assay, it is to be
understood that the nm23 antibody may be used in other assay
techniques, e.g., an agglutination assay wherein the nm23 antibody
is used on a solid particle such as a latex particle.
In accordance with another aspect of the present invention, there
is provided an assay kit or package for determining human nm23
protein which includes nm23 antibody, preferably nm23 antibody
elicited in response to nm23 protein. The nm23 antibody may or may
not be labeled with a detectable marker or label. If the kit is to
be used for an immunohistochemical assay, the kit may include
unlabeled nm23 antibody and a labeled antibody which immunobinds to
the nm23 antibody. If the kit is to be used in an immunoassay, the
kit may include both supported nm23 antibody and unsupported nm23
antibody which is preferably labeled with a detectable label or
marker. The kit may also include other components, such as buffers
etc.
The invention will be further described with respect to the
following examples; however, the scope of the invention is not to
be limited thereby. In the Examples, unless otherwise noted,
purifications, digestions and ligations are accomplished as
described in "Molecular Cloning, a laboratory manual" by Maniatis
et al. Cold Spring Harbor Laboratory (1982).
EXAMPLE 1
Two distinct cDNAs were isolated from a cDNA library made form
normal human fibroblast mRNA. Standard techniques were used
throughout. As a probe, we used the 502 base HpaII restriction
fragment of pnm23-M1. Steeg, et al. (3). This DNA was isolated from
agarose gel electrophoretograms using DE45 membrane (Schlicher and
Schuell). The DNA was made radioactive using the nick translation
reaction (Amersham kit) and 32PdCTP (Amersham). The individual
bacteria of the cDNA library, obtained from Hiroto Okayama,
(Okayama, et al. (4)) were dispersed on agarose luria broth plates.
Following growth they were transferred to nitrocellulose (Schlicher
and Schuell), lysed using 0.5 M NaOH and 1.5 M NaCl, and
neutralized in 1 M NH Ac. DNA was fixed to the nitrocellulose by
baking. Hybridization with the radioactive probe was conducted in
40% formamide, 0.75 M NaCl, 0.075 M Na citrate, 0.2% Bovine Serum
Albumin, 0.2% Ficol, and 0.2% polyvinyl pyrolidone, and 2 mg/ml
DNA. Hybridization was conducted for 15 hours at 42.degree. C.
Following hybridization, the filter was washed twice with 0.3 M
NaCl, 0.03 M Na citrate, at room temperature for 20 minutes
followed by two wastes at 42.degree. C. in 0.015 M NaCl and 0.0015
M Na citrate for 20 minutes each. Positive hybridization was
detected for 5 bacteria by autoradiography. These were purified by
single cell cloning.
DNA was extracted from each of the 5 clones and analyzed by
restriction enzyme analysis. A distinct pattern was identified for
two clones, pnm23-H1 and pnm23-H2. A second nm23-H2 cDNA clone was
isolated from a human lung cDNA library using pnm23-H2 as a probe,
the clone is termed nm23-H2S. The nm23-H1 and nm23-H2S clones were
subjected to further analysis.
The DNA sequence of pnm23-H1 and pnm23-H2s was determined using the
dideoxy chain termination method (5) (U.S. Biochemical kit). For
this purpose, the cDNA of pnm23-H1 and pnm23-H2S were removed from
the plasmid and inserted using standard techniques into the Sal I
site of M13mp18 (BRL). DNA sequence analysis was conducted using
synthetic 17 base oligonucleotides as reaction primers. The DNA
sequence of pnm23-H1 and pnm23-H2S is shown in FIG. 6.
FIG. 6 show that pnm23-H1 contains nucleotide sequence upstream of
the putative translation initiation codon (nucleotide 87). The
non-identity of nucleotide sequence (94% similarity) indicates that
pnm23-H1 and pnm23-H2S are the products of separate genes.
EXAMPLE 2
Production of nm23-H1 and nm23-H2S Protein
The nucleotide sequence of pnm23-H1 and H2S can be translated into
a predicted protein sequence for the corresponding proteins.
Several methods can be used to generate such protein.
(1) Standard chemical procedures can be used to synthesize peptides
corresponding to all or a portion of the nm23-H1 or H2S amino acid
sequence. These peptides can be coupled to carrier proteins such as
KLH for antibody production.
(2) Protein corresponding to all or part of nm23-H1 or part of
nm23-H2S can be synthesized in bacteria under the direction of
bacterial transcription promotion signals. The nm23-H1 protein has
been expressed under direction of the bacteriophage lambda PL
promoter in a vector similar to others described (6). This vector
was constructed as shown in FIG. 1. The plasmid pBR322 was digested
with EcoRI and AvaI and the base fragment isolated by agarose gel
electrophoresis. This was mixed with a synthetic restriction
fragment (FIG. 1) containing several enzyme sites, a bacterial
ribosome binding site and a translation initiation codon containing
a NcoI restriction enzyme site. These were reacted with T4 DNA
ligase transformed into E.coli and plasmids of correct structure
identified. DNA from these plasmids was digested with BstXI and
BamHI and mixed with a BstXI-BglII digestion of the 4.5 kb Hind III
fragment of bacteriophage DNA. This BstXI-BglII fragment contains
the PL promoter. Following ligation and transformation into E. coli
(which contains a cI 857 prophage) plasmids containing the
structure shown in FIG. 1 were identified. DNA from these plasmids
was digested with BstXI and HpaI and the cohesive ends of each DNA
filled in by E. coli DNA polymerase I large fragment. This was
ligated using standard conditions and transformed into E. coli (7).
The nm23-H1 was removed from M13mp18 by digesting with NcoI at a
ratio of 1 unit of enzyme per 1 .mu.g DNA for 2 minutes to produce
partially digested molecules as verified by the conversion of
supercoiled molecules to linear forms. This was phenol/CHCl.sub.3
extracted and ethanol precipitated to remove NcoI enzyme and
further digested with EcoRI. The 0.7 kb fragment was isolated from
agarose gel electrophoretograms. This fragments were combined with
plasmid pPL which had been digested with EcoRI and NcoI ligated and
transformed into E. coli.
Bacterial clones were identified which could direct the synthesis
of human nm23-H1 protein. Bacteria were grown to OD 660=1 at
32.degree. C. and temperature shifted to growth at 42.degree. C.
for 16 hours. Total bacterial protein was examined by
electrophoresis in 15% polyacrylamide gels containing SDS. The
human nm23-H1 protein was identified as a 19 kDa protein, capable
of reacting with anti-peptide antisera directed against amino acids
86 to 102 of the protein.
The human nm23-H1 protein can be purified from the bacteria by a
variety of methods. For example, following growth and temperature
shift induction bacteria were lysed by sonication in 20 mM Tris pH
7.5 150 nM NaCl (TBS). Insoluble material was removed by
centrifugation at 100,000.times.g for 30 minutes. Ammonium sulfate
was then added to 40% saturated solution and proteins allowed to
precipitate at 4.degree. C. for 10 minutes. These proteins were
removed by centrifugation at 100,000.times.g for 10 minutes. Solid
ammonium sulfate was added to 60% saturated solution and proteins
allowed to precipitate for 1 hr. at 4.degree. C. These proteins
were collected by centrifugation at 100,000.times.g and the
precipitate dissolved in TBS. Following dialysis for 16 hours, a
fine precipitate is collected by centrifugation at 10,000.times.g
for 10 minutes. This is made soluble in TBS and 1 mM DTT. Protein
prepared in this way is more than 80% pure as judged by SDS
polyacrylamide gel electrophoresis. Protein prepared in this way is
suitable for use as an immunizing antigen in antibody production
and in biological modification experiments.
The nucleotide sequence of nm23-H1 and H2S allow the expression of
either protein in eucaryotic cells. There are a variety of systems
available for expression of proteins in cells ranging from yeast to
human tissue culture cells. The essential elements required for
expression of nm23-H1 or H2S protein were nucleotide sequences
capable of directing synthesis of nm23.
EXAMPLE 3
Production of nm23 Antibody
The products described in Example 2 section can be used as
antigens. These can be used intact or following coupling to a
carrier protein such as Keyhole Limpex Hemocyanin. Coupling can be
conducted using established techniques and using such crosslinking
agents as EDC. The antigen is then mixed with adjuvant (e.g.,
Freund's) and injected into the animal (such as rabbit, rat, or
goat). Following booster injections with antigen mixed with
adjuvant (e.g., Freunds incomplete) the animal is bled and sera
prepared. The presence of antibody can be monitored by
immunoprecipitation, western blot, or solid phase binding assay
(e.g., ELISA). Polyclonal antisera to nm23-H1 or H2S can be
prepared in purified form by affinity chromatography. The
immunoglobulin molecules can be obtained from the sera by
staphylococcal protein A binding and anti-nm23-H1 or H2S obtained
by binding to a solid matrix to which the appropriate nm23 antigen
has been chemically fixed.
EXAMPLE 4
Preparation of Monoclonal Antibody
Balb/c mice were made immune by 3 IP injections of 100 ug purified
nm23-H1 protein of 1 week intervals mixed with Freund's complete
adjuvant for the immunization and Freund's incomplete adjuvant for
the boosters. Hybridomas were prepared by the method of Lane et al.
Methods in Enz. 121, p. 183 (1986). The fusion partner was the
myeloma P3x63-Ag8.653 obtained from ATCC. Fused cells were plated
with intraperitoneal cells obtained by the method of Lane, et al.,
Hybridoma, Vol. 7 p. 289 (1988). Hybridomas were grown in DMEM
supplemented with NCTC-109 (Gibco) 7.5% Fetal Bovine Serum (Sigma)
7.5% CSPR-3 (Sigma) 1 mM Na pyruvate, 100 units Penicillin, 100 ug
streptomycin, 10 ug/ml insulin, and 25 uM B-mercapto ethanol,
containing 0.1 mM hypoxanthine, 0.4 uM amphoterin, and 0.016 mM
Thymidine. Hybridoma clones were grown in 96 well dishes for two
weeks. Anti-nm23 producing hybridomas were identified by ELISA.
Purified nm23-H1 protein was attached to Immulon 1 dishes and
hybridoma culture media were allowed to react for 2 hours at room
temperature. Antibody reaction was detected using biotinylated goat
anti-mouse antibodies and streptavidin horseradish peroxidase using
the conditions in the BRL HyBRL kit. Positive hybridomas were
cloned by limiting dilution in the above media containing 5%
hybridoma growth supplement (Fisher). These were tested for
reactivity in ELISA. This experiment has resulted in the isolation
of two monoclonal antibodies identified as nmE302 and nm102B.
EXAMPLE 5
Detection of Metastatic Tumors
Antibodies specific for the human nm23-H1 and H2S proteins can be
made as described. One such antibody directed against amino acids
45 to 61 of the nm23-H1 sequence was used to detect nm23 protein in
tumor sections. Standard techniques can be used for the preparation
of sections for immunohistochemistry. These methods include frozen
sections or formalin fixation of the sample followed by paraffin
embedding. In this example, tumor sections were fixed overnight in
10% neutral buffered formalin and
embedded in paraffin using an automatic tissue processor (Fisher).
Five micron sections were cut and deparaffinized using standard
procedures involving xylenes and alcohol. Sections were then
immunostained using affinity purified anti-peptide antibody at
1/200 dilution: Immunostaining was done using standard techniques
as provided by the manufacturer (Vector) using biotinylated goat
antirabbit antibody followed by avidin biotinylated horse radish
peroxidase. The color reaction (diamino benzidine tetrahydro
chloride) at 0.5 mg/ml for 5 minutes at room temperature was
followed by a water wash to stop reaction. Sections were then
counter stained using Mayers hematoxylin, dehydrated and coverslips
applied using standard methods. Sections were then examined by
light microscopy for distinct cytoplasmic staining. Two samples
from breast cancer patients where tumor had spread to the axillary
lymph nodes show little staining; two samples from patients with
cancer confined to the breast show distinct staining. This
indicates that detection of low nm23 protein expression identifies
malignant tumors with a propensity to spread outside the primary
site.
The following is an example of a scoring system which has proved
effective in the ability to score primary breast tumors as high or
low level nm23 staining cells.
1) Examination of tumors using 10.times. objective and location of
regions with the largest percentage of weakly staining cells.
2) Examination of the regions found in step 1 under high power
(40.times. objective) and determination of the percentage of weakly
staining cells, by counting the cells.
3) Evaluation of the percentage of weakly staining cells, should
the percentage of weakly staining cells exceed 35% the tumor is
considered to have low nm23 staining.
This scoring system has been used in distinguishing between a group
of patients with low nm23 expression and a poorer overall survival
rate and those patients with high nm23 expression and a higher
overall survival rate. FIG. 2, is a graph depicting tumor cell
percentage of nm23 staining vs estimated survival in years. In
FIGS. 2A, 2B and 2C, are determinations made by evaluations of the
same data set by three independent pathologists. The dashed lines
represent tumors expressing high nm23 expression and the solid
lines indicate low nm23 expression.
EXAMPLE 6
Somatic Cell Hybrid Analysis of Chromosal Localization
Somatic cell hybrid analysis of chromosamal localization. The
isolation and characterization of the hybrids has been described
(9-10). DNA samples from independent human-mouse and human-hamster
somatic cell hybrids and subclones were digested with EcoRi, and
the fragments resolved on 0.7% agarose gells. Southern blots were
prepared on nylon membranes and hybridized to a random primer
labeled 756 bp nm23-h1 cDNA insert (18). Blots were washed at high
stringency (<10% divergence) in 0.1.times.SSC.sup.3, 0.2% (w/v)
SDS at 55.degree. C. After autoradiography, the presence of the
hybridizing human sequences in the DNA samples was correlated with
the specific human chromosomes retained in each of the somatic cell
hybrids.
The nm23-H1 gene was localized to human chromosome 17 by Southern
blot analysis of DNA samples isolated from human-rodent somatic
cell hybrids (FIG. 7). In EcoRl digests the 21 kb and 4.6 kb (or
2.2 kb and 2.4 kb alleles) hybridizing human sequences segregated
concordantly with chromosome 17 and discordantly (greater than 29%)
with all other human chromosomes. A 1.7 kb human sequence
segregated with chromosome 16 in these hybrids. In a second set of
cell hybrids, in which human fiborblasts containing a 17;22
(p13;q11) reciprocal chromosome translocation were fused with
Chinese hamster cells (9), the nm23-H1 gene segregated with the
17p12-qtr translocation chromosome and discordantly with the 17p13
band (data not shown). Thus, the nm23-H1 gene and the p53 tumor
suppressor gene at 17p13 (10) were localized to different regions
of chromosome 17.
EXAMPLE 7
In Situ Hybridization to Metaphse Chromosomes
Peripheral blood lymphocytes from a healthy male (46;XY) were
cultured for 72 h at 37.degree. C. in RPMI-1640 supplemented with
15% fetal bovine serum, phytohemagglutinin (0.5 ug/ml), and
antibiotics. Cultures were then synchronized by addition f BudR
(100 ug/ml) for 16 h prior to washing and resuspension in fresh
medium containing thymidine (2.5 ug/ml) and incubation for an
additional 5.5 h (11) with Colcemid (0.05 ug/ml) present during the
final 20 min. The cells were centrifuged, swollen, and fixed. Air
dried metaphase spreads were prepared by standard procedures (12).
After treatment with RNAse A (100 ug/ml) for 1 h at 37.degree. C.,
the chromosomal DNA was denatured for 3 min in NaOH (0.07 N) in
ethanol (64%) (13-14). Radiolabeled probe (specific activity
3.2.times.10.sup.7 cpm/ug) was prepared by nick translation of the
pNM23-H1 plasmid DNA with [.sup.3 H]dTTP and [.sup.3 H]dCTP. the
probe was mixed with hybridization solution (formamide, 5% dextran
sulphate, 2.times.Denhardt's solution, 2.times.SSC, 5 mM EDTA, 20
mM sodium phosphate (pH 6.4), and 200 ug/ml sheared herring sperm
DNA), heat denatured, applied to slides (3.times.10.sup.5 cpm
probe/slide), and hybridized for 20 h at 42.degree. C. to remove
the non specifically bound probe and coated with a 50% solution of
NTB2 nuclear track emulsion (Kodak, Rochester, N.Y.). The slides
were stored dessicated at 4.degree. C. for 9 days and then
developed, stained (0.5% Wright's stain) and photographed. The
slides were destained and chromosomal banding was obtained by
staining with Hoechst 33258 (150 ug/ml) for 30 min and exposure to
UV illumination for 30 min after rinsing. The slides were again
stained with Wright's stain and the same metaphase spreads were
rephotographed (120).
The nm23-H1 gene was further regionally localized to the
centromeric region of chromosome 17 (p11-q11) by in situ
hybridization to metaphase chromosomes (FIG. 3), and a cross
hybridizing sequence on chromosome 16 was also observed (data not
shown).
The functional nm23-H1 gene was definitely assigned to chromosome
17 by two different methods: first, a 200-base pair probe prepared
from the 3' untranslated sequence from this cDNA identified the 21
kb EcoRI band which segregated with chromosome 17, and detected no
sequences on chromosome 16 (data not shown); and second, using both
EcoRl and Bg1 II polymorphisms for linkage analysis in the 40
C.E.P.H. pedigrees (15), a highly significant linkage was observed
to the Hox-2 marker assigned to chromosome 17, which also suggested
a regional localization to the proximal portion of the long arm, at
17q21. This is from a manuscript of STEEG's which is under
preparation entitled: Chromosomal localization of Human NM23-H1 in
the C.E.P.H. data base.
EXAMPLE 8
Allelic Deletion
Genomic DNA was isolated from the normal and tumor tissues of 109
cancer patients by standard methods. DNA was restricted with Bgl
II, resolved on 0.8% agarose gels, and Southern blots were
prepared. Southern blots were hybridized to a random primer labeled
756-base pair pNM23-H1 insert (18) in 50% (v/v) formamide,
5.times.SSC, 50 mM Tris-HCI, (pH/7.5), 5.times.Denhardt's solution,
0.5% (w/v) SDS, 250 ug/ml denatured salmon sperm DNA, 0.1% (w/v)
dextran sulfate at 42.degree. C., washed to a final stringency of
0.1.times.SSC, 0.2% (w/v) SDS-1, mM EDTA, 65.degree. C.; and
hybridization detected by autoradiography.
A Bgl II restriction fragment length polymorphism (RFLP) of human
chromosomal DNA, which identified nm23-H1 allelic bands at 2.3 kb
and 7.6 kb, was used for analysis of possible nm23-H1 somatic
allelic deletion in human carcinomas. A total of 109 paired DNA
samples from matched normal tissue and renal, lung, colon or breast
carcinomas were analyzed for possible nm23-H1 allelic deletions
(FIGS. 4-5). In human breast carcinomas, 64% of informative
(heterozygous) tumors exhibited a deletion of one nm23-H1 allele
(FIG. 4A). Previous studies with this same cohort of breast tumors
analyzed allelic deletion at the transforming growth factor.alpha.
(2p13), somatostatin (3p28), MYB (6q22-23) and platelet derived
growth factor (22q12.3-q13.1) loci, and reported a background rate
of allelic deletion of less than 7% (16). In non-small cell lung
carcinomas, 42% of informative cases exhibited nm23-H1 allelic
(FIG. 4B). All of the lung tumors exhibiting nm23-H1 allelic
deletions were adenocarcinomas; tumors without detectable nm23-H1
allelic deletion included adenocarcinomas, osteosarcomas, squamous
cell carcinomas and large cell carcinomas. These data stand in
contrast to previous studies in non-small cell lung carcinomas, in
which allelic deletions at other chromosome 17 loci were observed
primarily in squamous cell carinomas (17). Among renal carcinomas
from patients were metastatic disease, 20% of informative cases
exhibited nm23-H1 allelic deletion (FIG. 4C). A cell line
established from each tumor to eliminate normal cell contamination
indicated that the small amount of remaining nm23-H1 hybridization
to tumor DNA was due to the presence of contaminating normal cells.
Finally, among invasive (Duke's C classification) colon carcinomas,
22% of informative cases exhibited nm23-H1 allelic deletions (FIG.
5, normal and tumor lanes). The data establish that the nm23-H1
gene is subject to somatic allelic deletion in human tumors.
In the colon carcinoma case shown in FIG. 5, DNA samples from
normal colonic mucosa, the primary tumor and a lymph node metatasis
were examined. In addition to the allelic deletion in the primary
tumor, previously described, a homozygous deletion of nm23-H1 was
observed in the lymph node metastasis. Rehybridization of the same
filter with a control Ha-ras probe (11p15.5) indicated
approximately equivalent amounts of DNA in each lane (FIG. 5). On a
long exposure, a small amount of hybridization to the nm23-H1 bands
was observable in the lymph node metastasis DNA, but may result
from contaminating normal cells, as was demonstrated in renal
carcinoma. The data in this case indicate a sequential series of
alterations, from a single allelic deletion to a homozygous
deletion, that was correlated with metastatic progression. The
normal, primary tumor and lymph node metastasis DNAs from this
patient each exhibited bands of hybridization to the p53 suppressor
gene on Southern blots, but the case was uninformative for allelic
deletion at this locus (data now shown). Thus, the nm23-H1
homozygous deletion data were not due to the complete deletion of
both copies of chromosome 17.
The relative independence of nm23-H1 allelic deletions to allelic
deletions at other chromosome 17 loci was determined. The
normal/tumor DNA sets were hybridized to at least three other
chromosome 17 probes, including p53, (17p13, Bgl II digest);
YNZ22.1, (17p13.3, BamH1, Taq1 or Hin fl digests); p144D6 (17p13.3,
Pstl digest); pHF 12.2 (17p12, Mspl digest); THH59 (17q23-25.3,
Pvull digest). Nine normal/tumor DNA sets were identified that: (a)
were informative for both nm23-H1 and another chromosome 17 probe,
and (b) exhibited a nm23-H1 allelic deletion. Of these, 2 cases
exhibited an nm23-H1 allelic deletion, but were heterozygous at the
YNZ22.1 locus; one case exhibited an nm23-H1 allelic deletion, but
was heterozygous at the p53 locus. The data indicate that deletions
of relatively large areas of chromosome 17 occur in many tumors,
suggesting that nm23-H1 and/or other chromosome 17 genes may be the
targets; in 3/9 cases, however, evidence for specificity in nm23-H1
allelic deletion was obtained.
Numerous modifications and variations of the present invention are
possible in light of the above teachings; therefore, within the
scope of the appended claims, the invention may be practiced
otherwise than as particularly described.
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